Effects Of Helicobacter Pylori Virulence Factor-CagA And Urease Metabolite-NH₄⁺ On Mucins In AGS Cells

Objective Helicobacter pylori infection is one of the major causes of human gastric carcinoma and can disturb the gastric mucosa barrier.Mucins have not only lubricating and protecting functions,but are also related to signal transduction,turnover of gastric epithelium and carcinogenesis of gastric mucosa.Studies have shown that gastric mucin expression changes in Helicobacter pylori infection,but little is known about the specific mechanism of Helicobacter pylori induced changes in mucin expression in gastric cancer cells.Therefore,we studied the effects of Helicobacter pylori virulence factor Cag A and urease metabolite NH4+on the expression of mucin in AGS cell line,so as to clarify the mechanism of gastric cancer and provide a basis for diagnosis and treatment of gastric cancer.Method Cag A overexpression plasmid was transfected into human gastric cancer AGS cell line,and AGS cells were divided into transfected Cag A plasmid(p CDNA3.1-Cag A)group,blank plasmid(p CDNA3.1)group and untransfected plasmid group(CK)group.Thenthe expression levels of Cag A m RNA and mucin MUC2,MUC5 AC,MUC6 and MUC5 B m RNA in three groups of cells were detected by q PCR,Western Blot was used to verify the expression of Cag A protein,and the expression of mucin,m RNA,and protein was detected by immunofluorescence.AGS cells and AGS cells transfected with Cag A plasmids were treated with 0,5,10,15,and20mmol/L ammonium chloride.The expression of mucin MUC2,MUC5 AC,MUC6,MUC5 B gene and protein were detected by quantitative PCR and immunofluorescence.Results1.In comparison,Cag A m RNA and Cag A protein were expressed only in p CDNA3.1-Cag A group,but no expression in CK and p CDNA3.1 group cells:p CDNA3.1-Cag A vs p CDNA3.1(P?0.05);p CDNA3.1-Cag A P(?0.05).2.The expression level of MUC5 AC,MUC2 and MUC5 B in group p CDNA3.1-Cag A was significantly higher than that in group p CDNA3.1(P < 0.05)and CK group(P< 0.01);p CDNA3.1-Cag A expression in MUC6 group was highter than that in group p CDNA3.1(< 0.01),similar to that in group A.(> 0.05).3.The expression of mucin MUC5 AC,MUC6 and MUC2 m RNA increased with the increase of NH4+concentration in AGS cells until 15mmol/L reached the peak value,and was significantly higher than AGS cells without ammonium treatment(P < 0.01;P < 0.01;P < 0.01).When the concentration of NH4+was 15mmol/L,the expression of MUC5 B m RNA was significantly higher than that of other NH4+concentrations(P < 0.05).4.The expression level of mucin MUC5 AC,MUC2 and MUC6 m RNA in the AGS cells transfected with Cag A at NH4+15mmol/L was significantly higher than0mmol/L(p < 0.01;p < 0.05;p < 0.05).The expression of MUC5 B m RNA increased with the increase of NH4+concentration,and was significantly higher than that of NH4+0 mmol/L(P < 0.01).5.Immunofluorescence analysis confirmed the above results.Conclusion1.Cag A up-regulates MUC5 AC,MUC2 and MUC5 B expression,but has no effect on MUC6 expression in AGS cells.2.Ammonium ions increase MUC5 AC,MUC2,MUC5 B,and MUC6 expression.3.Hp may affect the expression of MUC2,MUC5 AC,MUC5B and MUC6 in AGS cells via the toxicity factor Cag A and / or urease metabolite NH4+.